ABSTRACT Nitric oxide (NO) and cellular redox signaling are linked pathways crucially involved in the physiology and pathophysiology of the cardiovascular system. The main receptor for NO is soluble guanylyl cyclase (GC1), a heme-containing heterodimer. Upon binding of NO to the heme, GC activity is stimulated several hundred-fold to produce cGMP. Despite the critical role of the NO-cGMP pathway in vascular homeostasis and pathophysiology, the mechanisms of regulation and activation of GC1 are still poorly understood. We previously showed that S-nitrosation and other thiol oxidations of specific Cys of GC1 causes it to become desensitized to NO catalytic stimulation, a phenomenon of therapeutic importance. In fact, GC1 is one of the most sought-after targets for treatment of cardiovascular diseases, in particular to overcome NO resistance in vascular dysfunction (i.e., when exogenous NO cannot correct disrupted vascular reactivity). We discovered that GC1 interacts with thioredoxin 1 (Trx1) via a mixed disulfide exchange and this interaction appears to protect GC1 from desensitization to NO stimulation. Interestingly, the GC1-Trx1 complex was increased by inducing cellular thiol oxidation with Angiotensin II and S-nitrosocysteine treatments. Our most recent investigations reveal the presence of disulfide bonds in GC1 and indicate that these disulfide bonds are different between unstimulated (basal) and NO-stimulated conditions, suggesting that thiol/disulfide switches could be involved in the mechanism of activation of GC1. Based on novel biochemical, proteomic and structural evidence, we propose that the transition of GC1 from basal levels of catalysis (generation of cGMP in the absence of NO) to high rates of cGMP production in response to NO-heme binding is mediated by breaking of specific disulfide bonds and potential formation of different disulfide bonds to create and stabilize a highly active catalytic conformation. Moreover, we will explore the hypothesis that the interaction between Trx1 and GC1 is involved in the mechanism of activation/deactivation by facilitating the reduction of disulfide(s) of the high catalytic state to promote the return to basal state and sensitization to a new cycle of NO activation. Using a combination of cellular, biochemical, Molecular Dynamics simulation and Mass Spectrometry experiments, we will identify and determine the function of disulfide bonds in Aim1 and establish the mechanism and biological relevance of GC1 interaction with Trx1 in Aim2. The unifying idea behind this project is the concept that NO signaling in cardiovascular biology via the classical NO-cGMP pathway is dependent on reactive disulfide(s) of GC1, their redox modulation and their redox- dependent interaction with other proteins. The etiology of cardiovascular function and misfunction could very well depend on the ability to maintain proper balance of GC1 thiol/disulfide switches.